Selection of an analytical method for evaluating bovine serum albumin concentrations in pharmaceutical polymeric formulations

Bovine serum albumin (BSA) is a commonly used model protein in the development of pharmaceutical formulations. In order to assay its release from various dosage forms, either the bicinchoninic acid (BCA) assay or a more specific size-exclusion high performance liquid chromatography (SE-HPLC) method are commonly employed. However, these can give erroneous results in the presence of some commonly-used pharmaceutical excipients. We therefore investigated the ability of these methods to accurately determine BSA concentrations in pharmaceutical formulations that also contained various polymers and compared them with a new and compared with a new reverse-phase (RP)–HPLC technique. We found that the RP-HPLC technique was the most suitable method. It gave a linear response in the range of 0.5 -100 µg/ml with a correlation coefficient of 0.9999, a limit of detection of 0.11 µg/ml and quantification of 0.33 µg/ml. The performed ‘t’ test for the estimated and theoretical concentration indicated no significant difference between them providing the accuracy. Low % relative standard deviation values (0.8-1.39%) indicate the precision of the method. Furthermore, the method was used to quantify in vitro BSA release from polymeric freeze-dried formulations.

Reduction of severe bovine serum associated matrix effects on carboxymethylated dextran coated biosensor surfaces

Surface plasmon resonance (SPR) based biosensor technology has been widely used in life science research for many applications. While the advantages of speed, ruggedness, versatility, sensitivity and reproducibility are often quoted, many researchers have experienced severe problem of non-specific binding (NSB) to chip surfaces when performing analysis of biological samples Such as bovine serum. Using the direct measurement of the bovine protein leptin, present in bovine serum samples as a model, a unique buffering system has been developed and optimised which was able to significantly reduce the non-specific interactions of bovine serum components with the carboxymethyl dextran chip (CM5) surface on a Biacore SPR The developed NSB buffering system comprised of HBS-EP buffer, containing 0.5 M NaCl, 0.005% CM-dextran pH 9.0. An average NSB reduction (n = 20) of 85.9% and 87.3% was found on an unmodified CM5 surface and a CM5 with bovine leptin immobilised on the chip surface, respectively. A reduction in NSB of up to 94% was observed on both surfaces. The concentration of the constitutive components and pH of the buffer were crucial in achieving this outcome. (C) 2008 Elsevier B.V. All rights reserved.

Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay.

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems.

We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC50 of 0.79 ng mL-1 for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay.

Cross-reactivity data for a range of 11ß-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques.

Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis

The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered. These foods include bread and other bakery products, crisps, chips, breakfast cereals, and coffee. To date, the diminutive size of acrylamide (71.08Da) has prevented the development of screening immunoassays for this chemical. In this study, a polyclonal antibody capable of binding the carcinogen was produced by the synthesis of an immunogen comprising acrylamide derivatised with 3-mercaptobenzoic acid (3-MBA), and its conjugation to the carrier protein bovine thyroglobulin. Antiserum from the immunised rabbit was harvested and fully characterised. it displayed no binding affinity for acrylamide or 3-MBA but had a high affinity for 3-MBA-derivitised acrylamide. The antisera produced was utilised in the development of an ELISA based detection system for acrylamide. Spiked water samples were assayed for acrylamide content using a previously published extraction method validated for coffee, crispbread, potato, milk chocolate and potato crisp matrices. Extracted acrylamide was then subjected to a rapid 1-h derivatisation with 3-MBA, pre-analysis. The ELISA was shown to have a high specificity for acrylamide, with a limit of detection in water samples of 65.7 mu g kg(-1), i.e. potentially suitable for acrylamide detection in a wide range of food commodities. Future development of this assay will increase sensitivity further. This is the first report of an immunoassay capable of detecting the carcinogen, as its small size has necessitated current analytical detection via expensive, slower, physico-chemical techniques such as Gas or Liquid Chromatography coupled to Mass Spectrometry. (c) 2007 Elsevier B.V. All rights reserved.